Cholesterol Regulates Glucose-stimulated Insulin Secretion through Phosphatidylinositol 4,5-Bisphosphate

Membrane cholesterol modulates the ability of glucose to stimulate insulin secretion from pancreatic β-cells. The molecular mechanism by which this occurs is not understood. Here, we show that in cultured β-cells, cholesterol acts through phosphatidylinositol 4,5-bisphosphate (PIP2) to regulate actin dynamics, plasma membrane potential, and glucose-stimulated insulin secretion. Cholesterol-overloaded β-cells exhibited decreased PIP2 hydrolysis, with diminished glucose-induced actin reorganization, membrane depolarization, and insulin secretion. The converse findings were observed in cholesterol-depleted cells. These results support a model in which cholesterol depletion is coupled through PIP2 to enhance both plasma membrane Ca2+ influx from the extracellular space, as well as inositol 1,4,5-triphosphate-stimulated Ca2+ efflux from intracellular stores. The inability to increase cytosolic Ca2+ may be the main underlying factor to account for impaired glucose-stimulated insulin secretion in cholesterol-overloaded β-cells. Membrane cholesterol modulates the ability of glucose to stimulate insulin secretion from pancreatic β-cells. The molecular mechanism by which this occurs is not understood. Here, we show that in cultured β-cells, cholesterol acts through phosphatidylinositol 4,5-bisphosphate (PIP2) to regulate actin dynamics, plasma membrane potential, and glucose-stimulated insulin secretion. Cholesterol-overloaded β-cells exhibited decreased PIP2 hydrolysis, with diminished glucose-induced actin reorganization, membrane depolarization, and insulin secretion. The converse findings were observed in cholesterol-depleted cells. These results support a model in which cholesterol depletion is coupled through PIP2 to enhance both plasma membrane Ca2+ influx from the extracellular space, as well as inositol 1,4,5-triphosphate-stimulated Ca2+ efflux from intracellular stores. The inability to increase cytosolic Ca2+ may be the main underlying factor to account for impaired glucose-stimulated insulin secretion in cholesterol-overloaded β-cells. In pancreatic β-cells, glucose-stimulated insulin secretion (GSIS) 2The abbreviations used are: GSISglucose-stimulated insulin secretionCholsoluble cholesterol (cholesterol-MβCD complex)IP3inositol 1,4,5-triphosphateKSISKCl-stimulated insulin secretionMβCDmethyl-β-cyclodextrinPIP2phosphatidylinositol 4,5-bisphosphatePMplasma membraneSMasesphingomyelinasePHpleckstrin homologyPLCδphospholipase CδGFPgreen fluorescent proteinHBSSHepes balanced saline solutionDiSBAC2(3)bis-(1,3-diethylthiobarbituric acid)trimethine oxonolDiIDiIC18(3) 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate. is mediated by effects on both membrane potential and cortical actin (1Wang Z. Thurmond D.C. J. Cell Sci. 2009; 122: 893-903Crossref PubMed Scopus (262) Google Scholar). F-actin exists as a dense web beneath the β-cell plasma membrane (PM), which inhibits the recruitment and sustained exocytosis of insulin secretory granules (2Orci L. Gabbay K.H. Malaisse W.J. Science. 1972; 175: 1128-1130Crossref PubMed Scopus (281) Google Scholar, 3Howell S.L. Tyhurst M. Diabetes Metab. Rev. 1986; 2: 107-123Crossref PubMed Scopus (27) Google Scholar). Disruption of the cortical F-actin network permits access of insulin granules to the cell periphery and enhances GSIS (4Thurmond D.C. Gonelle-Gispert C. Furukawa M. Halban P.A. Pessin J.E. Mol. Endocrinol. 2003; 17: 732-742Crossref PubMed Scopus (145) Google Scholar). In both primary and cultured β-cells, glucose induces F-actin remodeling to mobilize insulin granules for docking and fusion at the PM (5Nevins A.K. Thurmond D.C. Am. J. Physiol. Cell Physiol. 2003; 285: C698-C710Crossref PubMed Scopus (117) Google Scholar, 6Tomas A. Yermen B. Min L. Pessin J.E. Halban P.A. J. Cell Sci. 2006; 119: 2156-2167Crossref PubMed Scopus (131) Google Scholar). glucose-stimulated insulin secretion soluble cholesterol (cholesterol-MβCD complex) inositol 1,4,5-triphosphate KCl-stimulated insulin secretion methyl-β-cyclodextrin phosphatidylinositol 4,5-bisphosphate plasma membrane sphingomyelinase pleckstrin homology phospholipase Cδ green fluorescent protein Hepes balanced saline solution bis-(1,3-diethylthiobarbituric acid)trimethine oxonol DiIC18(3) 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate. The exocytosis of peripheral insulin granules is controlled by ATP-sensitive K+ (KATP) channels. In β-cells, KATP channels consist of four inwardly rectifying K+ channel (Kir6.2) subunits and four SUR1 (sulfonylurea receptor 1) subunits (7Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (621) Google Scholar). KATP channels are highly sensitive to changes in the ATP/ADP ratio, coupling glucose metabolism to membrane potential. Glucose stimulation increases the ATP/ADP ratio, inhibiting KATP channel activity to cause membrane depolarization, voltage-gated Ca2+ influx, and initiation of insulin secretion (8Ashcroft F.M. Gribble F.M. Diabetologia. 1999; 42: 903-919Crossref PubMed Scopus (381) Google Scholar). Impaired insulin secretion from pancreatic β-cells contributes to the progression of type 2 diabetes. Yet a full understanding of how GSIS normally occurs and how it is defective in diabetes has remained elusive (9Lyssenko V. Almgren P. Anevski D. Perfekt R. Lahti K. Nissén M. Isomaa B. Forsen B. Homström N. Saloranta C. Taskinen M.R. Groop L. Tuomi T. Diabetes. 2005; 54: 166-174Crossref PubMed Scopus (278) Google Scholar). Recently, it has been suggested that alterations in cellular cholesterol levels may play a critical role to modulate GSIS. The idea that excess cholesterol inhibits GSIS is supported by data from mice lacking the ABCA1 cholesterol transporter specifically in β-cells (10Brunham L.R. Kruit J.K. Pape T.D. Timmins J.M. Reuwer A.Q. Vasanji Z. Marsh B.J. Rodrigues B. Johnson J.D. Parks J.S. Verchere C.B. Hayden M.R. Nat. Med. 2007; 13: 340-347Crossref PubMed Scopus (337) Google Scholar). Islets from these mice have increased cholesterol and impaired GSIS. Similarly, elevated islet cholesterol levels in ApoE-deficient mice inhibit GSIS (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). Direct manipulation of membrane cholesterol in cultured β-cells further shows that excess cholesterol impairs GSIS, whereas cholesterol depletion enhances GSIS (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). Together, these data suggest that membrane cholesterol content contributes to the regulation of GSIS. The mechanisms by which cholesterol influences GSIS are not known. In addition to the ATP/ADP ratio, phosphatidylinositol 4,5-bisphosphate (PIP2) modulates the activity of KATP channels in β-cells (12Lin C.W. Yan F. Shimamura S. Barg S. Shyng S.L. Diabetes. 2005; 54: 2852-2858Crossref PubMed Scopus (42) Google Scholar). PIP2 is a minor phospholipid that regulates cytoskeletal organization, membrane trafficking, the generation of second messengers, and the function of ion channels (13Di Paolo G. De Camilli P. Nature. 2006; 443: 651-657Crossref PubMed Scopus (2080) Google Scholar). PIP2 is suggested to be important to GSIS, yet little is known about how this lipid is regulated in pancreatic β-cells. It has been shown in other cells that cholesterol controls PIP2 distribution in membranes and regulates its downstream effects in a cell type-dependent manner (14Epand R.M. Biochim. Biophys. Acta. 2008; 1778: 1576-1582Crossref PubMed Scopus (132) Google Scholar). In the present work, we tested the hypothesis that cholesterol regulates GSIS through PIP2. In cultured β-cells, we found that depleting cholesterol stimulates PIP2 hydrolysis, whereas overloading cholesterol enhances PM PIP2 accumulation. Changes in PM PIP2 in turn affect actin dynamics at the cell periphery and the extent of glucose-stimulated membrane depolarization in β-cells. Together with other data, we propose a model in which cholesterol acts through PIP2 to regulate both the actin cytoskeleton and plasma membrane potential, thus impacting multiple aspects of GSIS. 832/13 INS-1 β-cells were a kind gift from Dr. Christopher B. Newgard (Duke University, Durham, NC). The cells were cultured in RPMI 1640 with 11.1 mm d-glucose supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 mm Hepes, 2 mm l-glutamine, 1 mm sodium pyruvate, and 50 μm β-mercaptoethanol at 37 °C and 5% CO2 in a humidified atmosphere (15Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (710) Google Scholar). The pleckstrin homology (PH) domain of PLCδ fused to GFP (PHPLCδ-GFP) was a gift from Dr. Pietro De Camilli (Yale The cells were with and to the and cultured for to cells were by INS-1 cells with kind gift from Dr. University, M. C. J. 2003; PubMed Scopus Google Scholar). as well as insulin secretion were in Hepes balanced solution mm mm mm mm mm Hepes, mm mm and bovine (15Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (710) Google Scholar). insulin were from the cholesterol bis-(1,3-diethylthiobarbituric acid)trimethine oxonol and solution were from PIP2 and its and were from cholesterol of cholesterol of sphingomyelinase from and other were from of PIP2 to β-cells was as T. P. J. Cell 2005; PubMed Scopus (132) Google Scholar). PIP2 was with to of and 100 to the at for 10 by a were with this in the of for and and with the of this was cells the was with and a and were used to and was through a for of and a for was a with a by were a and were a a and a and were used for from the of the PM of cells was as M. S. Sci. PubMed Scopus Google Scholar). cells were with with μm in at 37 °C for with and with on for cells were as with the of with for cells. were M. J. 2000; PubMed Scopus Google and by cell and the with the cells. The of from the and cells of was as the cells were from to mm glucose for by for 2 in 2 mm were to mm 10 mm 50 for whereas were in were and to 2 mm glucose mm for at 37 which was for insulin by insulin protein content was the protein secretion was to protein was the cholesterol as (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). of cells were for at that a was and the was the for cells. were used to and on the PM were used to at cell were to a of was to the cells on the of mm glucose and mm for 10 mm for and 10 mm were were with for with for and with in with for at cells were with for 2 at 37 by in at this cells were and cells were M. D. J. PubMed Scopus Google to actin and cells were with for at 37 the 10 of which a of the fluorescent membrane potential was The solution the of mm glucose was to the solution the of the cells were with in 2 mm glucose and in a solution the of and glucose with as the for cells were in the solution with is in membrane which are to a of to cholesterol to other membrane be methyl-β-cyclodextrin a cholesterol the of this by on GSIS in pancreatic and cultured β-cells (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). cholesterol is by further is to the role of membrane in GSIS. membrane regulates GSIS, of cholesterol were was used to membrane the of which is found in membrane F.M. R. PubMed Scopus Google Scholar). β-cells 1) were is the in the of and is a critical of membrane in cells results in lipid effects on cholesterol M. C. J. 2003; PubMed Scopus Google Scholar). shown in whereas cholesterol decreased cholesterol cholesterol GSIS was by both and to overloading the cells with GSIS as (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). These data suggest that a lipid GSIS. The from cells is it that membrane is as at GSIS as cholesterol the effects of these on PM organization, we the that membrane are in Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar). in not has been used as of membrane Biophys. 2008; PubMed Scopus Google Scholar). The of in cells used in was by a of cells PM was by a fluorescent lipid with for the M. S. Sci. PubMed Scopus Google Scholar). In with the of a of in the that the of the membrane was to as M. S. Sci. PubMed Scopus Google Scholar). In cells with and in cells was increase in the a in membrane is with the idea that cholesterol and are of of regulates lipid and on membranes R. D. Sci. PubMed Scopus Google Scholar, A. F.M. J. 1999; Scopus Google shows that a in to membrane in INS-1 cells. and to membrane regulate the actin cytoskeleton (14Epand R.M. Biochim. Biophys. Acta. 2008; 1778: 1576-1582Crossref PubMed Scopus (132) Google Scholar). the observed changes in membrane the actin β-cells were with and for F-actin PubMed Scopus Google and J. PubMed Google in which the cell at the of of intracellular are used to cell from and in as the to changes in the cortical actin β-cells F-actin with a cortical actin and content was in the of to F-actin increased glucose stimulation and was further by to enhance PM is in with data that cholesterol depletion enhances in J. S. D. L. M. Sci. 2003; PubMed Scopus Google Scholar). The in F-actin was at the cell periphery to to the cell cholesterol-overloaded cells increased F-actin and These were in cells and found in glucose-stimulated cells. In cholesterol-overloaded by F-actin at the cell was the cellular cholesterol and F-actin cholesterol-overloaded β-cells were with both and a cholesterol J. B. J. Cell Google Scholar). shown in cells with a cholesterol content and the of of and in cells a of of cholesterol and F-actin was to a of cholesterol was present in membranes of intracellular to that observed in other cell M. D. J. PubMed Scopus Google Scholar, L. D. J. 2008; 49: PubMed Scopus Google Scholar, M. P. J. Mol. 2007; PubMed Scopus Google Scholar, J. Z. G. G. N. S. J. J. 2008; PubMed Scopus Google Scholar, N. R. Mol. PubMed Scopus Google Scholar, R. N. J. Mol. 2000; PubMed Scopus (278) Google Scholar, D. A. M. J. PubMed Scopus Google Scholar). in cells with PM in cholesterol F-actin how changes in the actin cytoskeleton GSIS. Disruption of F-actin by GSIS, whereas of F-actin by GSIS These results are with data that actin is for insulin secretion (4Thurmond D.C. Gonelle-Gispert C. Furukawa M. Halban P.A. Pessin J.E. Mol. Endocrinol. 2003; 17: 732-742Crossref PubMed Scopus (145) Google Scholar, PubMed Scopus Google Scholar, Z. Thurmond D.C. J. 2008; PubMed Scopus Google Scholar). Together, the data suggest that of membrane and increased membrane and GSIS by cortical actin with the idea that actin web may present a to insulin exocytosis (2Orci L. Gabbay K.H. Malaisse W.J. Science. 1972; 175: 1128-1130Crossref PubMed Scopus (281) Google Scholar). overloading the cells with cholesterol and the F-actin network at the cell periphery 2 and to of GSIS which on cholesterol for its and is important of actin (14Epand R.M. Biochim. Biophys. Acta. 2008; 1778: 1576-1582Crossref PubMed Scopus (132) Google Scholar, Mol. PubMed Scopus Google Scholar). not other PIP2 is how cholesterol may regulate PIP2 metabolism is not well J.M. J. PubMed Scopus Google Scholar, J. H. J. K. J. 2005; PubMed Scopus Google Scholar). PIP2 a GFP fused to the domain of PLCδ (PHPLCδ-GFP) was The domain PIP2 specifically and with and to the has been used to PIP2 metabolism S. T. PubMed Google Scholar, C. J. J. Cell 2003; PubMed Scopus Google Scholar). of from that decreased at the PM and increased its in the inositol 1,4,5-triphosphate in the this of is as of PM PIP2 to cytosolic as J. S. D. L. M. Sci. 2003; PubMed Scopus Google Scholar). with of cause cells to was that the increase in intracellular may be to the that addition was and with as little as 1 mm that these was in β-cells. is by the that was observed in distribution in that as a of factor addition J.M. T. J. Cell Sci. PubMed Google Scholar). soluble cholesterol was cells PM in and cytosolic was decreased The cells membrane to in cholesterol-overloaded cells and PIP2 at the PM from both depletion to other and the of PM PIP2 cholesterol overloading from PIP2 cytosolic was not and at the Glucose PIP2 hydrolysis, as S. A. A. Diabetes. 2007; 56: PubMed Scopus (42) Google yet this was and that by cholesterol depletion cells were with glucose and with the glucose-induced cytosolic was a and of from PM to cells were with and was observed cells were with soluble cholesterol second glucose was to stimulate PIP2 hydrolysis, as by a of increase in the was the stimulation was of was observed Together, these data support the that excess cholesterol inhibits glucose-stimulated PIP2 hydrolysis, and this be by cholesterol PIP2 regulates actin by with P.A. Nat. Rev. Mol. Cell PubMed Scopus Google Scholar). In PIP2 to for Nature. PubMed Scopus Google Scholar). PIP2 is to the of cholesterol on the actin the results in to effects of cholesterol on cell which may changes in the actin The PM of β-cells was to actin to PIP2 and the addition are shown in and in of β-cells was observed a of cells were with which and PIP2 A. K. G. S. S. Biochim. Biophys. Acta. 2000; PubMed Scopus Google was These data suggest that effects of cholesterol depletion on cell are at in by PIP2. soluble cholesterol was to the changes were of membrane of and and and of cell and and and The membrane as the β-cell cholesterol of these actin PubMed Scopus Google Scholar, C. Cell PubMed Scopus Google Scholar). These were by the addition of and results were were used in of not as cholesterol in the on a These data are in with the idea that recruitment of the of membrane in both cholesterol and PIP2 T. K. M. T. D. P. J. Cell 2000; PubMed Scopus Google Scholar). In the and a of cholesterol on cellular actin network in β-cells. further the and we at the distribution of PM PIP2 and cortical actin in β-cells. for as and PIP2 was present by on the PM of β-cells is with findings in other cell T. K. M. T. D. P. J. Cell 2000; PubMed Scopus Google Scholar, J. K. Mol. 13: PubMed Scopus Google Scholar). PIP2 by overloading β-cells with soluble cholesterol and that PIP2 T. K. M. T. D. P. J. Cell 2000; PubMed Scopus Google and were these of the cells were and cells were M. D. J. PubMed Scopus Google to of actin with in cells. with was shown to and T. P. J. Cell 2005; PubMed Scopus (132) Google Scholar). In of actin and PIP2 was observed in cholesterol-overloaded cells further the of PIP2 in actin dynamics, we to to inhibit PIP2 T. K. M. T. D. P. J. Cell 2000; PubMed Scopus Google and of PIP2 to increase PM PIP2 T. P. J. Cell 2005; PubMed Scopus (132) Google Scholar). actin at the cell periphery the effects of and soluble cholesterol on actin were by the of shows that of β-cells with the ability of to cortical actin 2 and In addition to the actin PIP2 KATP channels. PIP2 increases the and the of the KATP channel by with the T. D. S. T. B. Science. PubMed Scopus Google Scholar, S.L. Science. PubMed Scopus Google Scholar). the data membrane cholesterol as a of PM we the of cholesterol to modulate membrane potential in β-cells. used the which cells and in a manner that with the of membrane depolarization V. PubMed Scopus Google Scholar). In β-cells with glucose-stimulated membrane depolarization was and in β-cells with glucose was to stimulate membrane Together with the data that cholesterol to PM PIP2 these support and that PIP2 inhibits the ability of to KATP channels T. D. S. T. B. Science. PubMed Scopus Google Scholar). to β-cells membrane depolarization of cells in both 2 and mm this depolarization was that by glucose glucose-stimulated a to inhibit KATP channels PIP2 from cholesterol GSIS is increased in cholesterol-depleted cells in to membrane depolarization, KCl-stimulated insulin secretion be by membrane cholesterol is the of the KATP channel is in that the of PIP2 to regulate KATP channel activity insulin secretion to a in GSIS. with this cholesterol depletion and on on GSIS was regulation by cholesterol in with cholesterol depletion in GSIS cholesterol to GSIS stimulation not the of cholesterol to modulate insulin may be of KATP channels that to cholesterol regulation of GSIS, as (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). have shown important role of cholesterol to regulate secretory through of membrane G. H. Cell PubMed Scopus Google Scholar, Rev. Cell PubMed Scopus Google Scholar). the cellular mechanisms underlying this regulation are not understood. GSIS from pancreatic β-cells of regulated which mechanisms with other secretory that excess cholesterol impairs β-cell function and GSIS (10Brunham L.R. Kruit J.K. Pape T.D. Timmins J.M. Reuwer A.Q. Vasanji Z. Marsh B.J. Rodrigues B. Johnson J.D. Parks J.S. Verchere C.B. Hayden M.R. Nat. Med. 2007; 13: 340-347Crossref PubMed Scopus (337) Google Scholar, M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar, L.R. Kruit J.K. Verchere C.B. Hayden M.R. J. 2008; PubMed Scopus Google Scholar, M. 2009; 20: PubMed Scopus Google Scholar). The data suggest that membrane cholesterol regulates glucose-stimulated actin reorganization, membrane depolarization, and insulin secretion by PIP2 metabolism in β-cells. PIP2 distribution in the PM is cell and is on how PIP2 is (14Epand R.M. Biochim. Biophys. Acta. 2008; 1778: 1576-1582Crossref PubMed Scopus (132) Google Scholar, J. H. J. K. J. 2005; PubMed Scopus Google Scholar). of a of PIP2 with and the that PIP2 not to its T. N. A. S. T. J. 2005; PubMed Scopus Google Scholar, J.E. K. Z. R. R.M. J. 2006; PubMed Scopus Google Scholar, M.R. V. M. PubMed Google Scholar). model is that PIP2 is known as to (14Epand R.M. Biochim. Biophys. Acta. 2008; 1778: 1576-1582Crossref PubMed Scopus (132) Google Scholar). and as and PIP2 in a manner (14Epand R.M. Biochim. Biophys. Acta. 2008; 1778: 1576-1582Crossref PubMed Scopus (132) Google Scholar, T. K. M. T. D. P. J. Cell 2000; PubMed Scopus Google Scholar, R.M. P. S. J. PubMed Google Scholar, D. T. L. C. P. J. Cell 2000; PubMed Scopus Google Scholar, J. L. A. Biophys. J. 2008; PubMed Scopus Google Scholar). These the and the of the on and membrane cholesterol content G. J.M. G. 2009; PubMed Scopus Google Scholar). shown in cholesterol overloading may the of at the in the of PIP2 to the that of PIP2 at the T. K. M. T. D. P. J. Cell 2000; PubMed Scopus Google Scholar). is for PIP2 (14Epand R.M. Biochim. Biophys. Acta. 2008; 1778: 1576-1582Crossref PubMed Scopus (132) Google PIP2 is to for A. G. J. D. S. Biophys. J. PubMed Scopus Google Scholar, J. A. G. D. S. J. PubMed Scopus Google Scholar). it may be that by the of cholesterol depletion the of PIP2 for and enhances PIP2 as a the other changes to affect PIP2 the a in the is to account for the observed results to the of cholesterol on PIP2 activity is by cholesterol be in cholesterol depletion PM PIP2 it acts from that the of PIP2 for effects from in which PIP2 is by by in addition to PIP2 hydrolysis, glucose PIP2 J. PubMed Scopus Google Scholar). increase in PM PIP2 may be by the of the for PIP2 in R. J. Sci. PubMed Scopus Google Scholar, K. S. M. H. M. Science. 1999; PubMed Scopus Google Scholar). is that glucose-stimulated PIP2 is in cholesterol-depleted cells. The data cholesterol regulation of PM PIP2 with the role of PIP2 to the actin suggest that PIP2 in actin in β-cells. by PIP2 PM cholesterol regulates actin and depletion PM PIP2 and actin at the cell whereas cholesterol PM PIP2 and to the of cortical actin and of membrane The model we propose is with that increased PIP2 actin and that PIP2 to actin P.A. Nat. Rev. Mol. Cell PubMed Scopus Google Scholar). In of PIP2 results in increased and cell J. 2008; PubMed Scopus Google not the effects of cholesterol overloading we observed in β-cells The KATP channel intracellular metabolism to membrane potential in pancreatic β-cells. of KATP channels a in the ATP/ADP is in that to of the KATP channel P.A. A. J. Physiol. PubMed Scopus Google Scholar, S. P. F.M. J. Physiol. PubMed Scopus Google Scholar). as the to PIP2 cholesterol KATP channels sensitive to of PIP2 in the of excess cholesterol by of the channel and β-cell membrane depolarization in to model is with the that increased PIP2 a in GSIS by KATP channels to (12Lin C.W. Yan F. Shimamura S. Barg S. Shyng S.L. Diabetes. 2005; 54: 2852-2858Crossref PubMed Scopus (42) Google Scholar). that excess cholesterol inhibits GSIS (10Brunham L.R. Kruit J.K. Pape T.D. Timmins J.M. Reuwer A.Q. Vasanji Z. Marsh B.J. Rodrigues B. Johnson J.D. Parks J.S. Verchere C.B. Hayden M.R. Nat. Med. 2007; 13: 340-347Crossref PubMed Scopus (337) Google Scholar, M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google yet the molecular mechanisms underlying this have not been Here, we show that cholesterol-overloaded β-cells are to glucose stimulation in of PIP2 hydrolysis, actin reorganization, and membrane PIP2 effects on the of KATP channels to which of S.L. Science. PubMed Scopus Google Scholar). results how defective regulation of KATP channel activity may from excess mechanism may be important for the of type 2 it how increased cholesterol on β-cells to insulin secretion and glucose the and role of cholesterol in cellular it is that mechanism is for the observed in β-cells. it that intracellular Ca2+ may be a by which cholesterol modulates GSIS (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). data support the that cholesterol depletion increases PIP2 and which to the of Ca2+ from intracellular stores. PM PIP2 induces membrane depolarization by the of KATP which voltage-gated Ca2+ The of is to which PIP2 in pancreatic and β-cells A. Diabetes. 2005; 54: PubMed Scopus (42) Google Scholar, S. A. J. Cell Sci. 2005; PubMed Scopus Google Scholar, S. A. J. PubMed Scopus Google Scholar). The converse may in cholesterol-overloaded the intracellular Ca2+ to glucose stimulation (11Hao M. Head W.S. Gunawardana S.C. Hasty A.H. Piston D.W. Diabetes. 2007; 56: 2328-2338Crossref PubMed Scopus (236) Google Scholar). the regulation of PIP2 distribution and actin is cell the findings in β-cells may to other cells. The in the of KATP channels in has been suggested to from effects of PIP2 T. D. S. T. B. Science. PubMed Scopus Google Scholar). data further suggest that membrane cholesterol may modulate KATP channel in In for KATP channels are the of of P.A. A. J. Physiol. PubMed Scopus Google Scholar). membrane cholesterol regulates this as well as in other cells be the of Pietro De Camilli and for the Dr. Christopher Newgard for and Min and for used the for Cell and the Diabetes Cell at of with